Cassette for sample preparation

ABSTRACT

Apparatuses for preparing a sample are disclosed herein. The apparatuses include a chamber, a first valve at least partially disposed in the first chamber, a second valve at least partially disposed in the first chamber, and a pump comprising an actuator and nozzle.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/357,947, filed Jan. 25, 2012, entitled “CASSETTE FOR SAMPLEPREPARATION,” which is a continuation of U.S. application Ser. No.12/789,831, now U.S. Pat. No. 8,124,024, filed May 28, 2010, entitled“CASSETTE FOR SAMPLE PREPARATION,” which is a continuation of U.S.application Ser. No. 11/582,651, now U.S. Pat. No. 7,727,473, filed Oct.17, 2006, entitled “CASSETTE FOR SAMPLE PREPARATION,” which claims thebenefit of U.S. Provisional Application No. 60/728,569, filed Oct. 19,2005, entitled “METHOD AND APPARATUS FOR ISOLATING NUCLEIC ACID,” U.S.Provisional Application No. 60/753,622, filed Dec. 22, 2005, entitled“CASSETTE FOR SAMPLE PREPARATION,” and U.S. Provisional Application No.60/753,618, filed Dec. 22, 2005, entitled “CASSETTE FOR SAMPLEPREPARATION,” each of which is hereby incorporated by reference.

BACKGROUND

1). Field of the Invention

The present invention relates to the field of biotechnology devices and,in particular, to devices and methods for preparing samples.

2). Discussion of Related Art

DNA can be used to develop new drugs or to link someone to a crime.However, before this can be done, the DNA must be isolated from asample. These samples include, for example, blood, urine, human cells,hair, bacteria, yeast and tissue. Each of these samples include cells,which include nucleic acid. Nucleic acid is a nucleotide chain, whichconveys genetic information. The most common forms of nucleic acid areDNA and RNA.

In order to isolate the nucleic acid from the samples, prior art devicesuse a tray having several exposed cavities. The sample is placed intoone of the cavities and conventional processing steps are used toisolate the DNA from the sample.

This prior art system has several disadvantages, includingcontamination. Since the cavities are exposed, contaminants can easilyaffect the DNA. In addition, the prior art system requires thepreparation of several samples at one time. It is difficult to prepareone or two samples at a time using the prior art devices.

SUMMARY

A cassette for preparing a sample is disclosed herein. The cassetteincludes at least one mixing chamber for receiving a sample of cells; afirst holding chamber; an enzyme in the first holding chamber, theenzyme being transferable into the at least one mixing chamber to breakthe cells and release nucleic acid from the cells to create bulkmaterial and the nucleic acid in the bulk material; a second holdingchamber; magnetic particles in the second holding chamber, the magneticparticles being transferable to the at least one mixing chamber to bindwith the nucleic acid; and at least one magnet, positionable to attractthe magnetic particles together with the nucleic acid and at leastpartially separate the nucleic acid from the bulk material in the atleast one mixing chamber.

The mixing chamber has a top surface, the top surface having an openingtherein. A removable lid for accessing the opening of the mixing chamberis also provided. The enzyme may be proteinase K. First and secondplungers in the first holding chamber and second holding chamber mayalso be provided, each plunger being movable to transfer the enzyme andmagnetic particles, respectively, into the mixing chamber. A thin filmthat is breakable to transfer the enzyme and magnetic particles throughrespective ruptures in the thin film into the mixing chamber may also beprovided. First and second plungers in the first holding chamber andsecond holding chamber, each plunger being movable to break the thinfilm and transfer the enzyme and magnetic particles, respectively intothe mixing chamber, may also be provided. A third holding chamber and alysis solution in the third holding chamber may also be provided, thelysis solution being transferable into the mixing chamber to solubilizethe bulk material. A fourth holding chamber and a binding solution inthe fourth holding chamber may also be provided, the binding solutionbeing transferable into the mixing chamber to bind the nucleic acid tothe magnetic particles. A heating element for heating the mixing chambermay also be provided.

The cassette may also include a first separation piece having a surface;a first transfer piece having a surface with a cavity therein, the atleast one magnet transferring the magnetic particles together with thenucleic acid into the cavity in the surface of the first transfer piece,the first transfer piece being movable relative to the first separationpiece so that the magnetic particles together with the nucleic acid moveout of the mixing chamber and past the surface of the first separationpiece; and a first receiving chamber which receives the magneticparticles and nucleic acid after moving past the surface of the firstseparation piece. The cassette may further include a second separationpiece having a surface; a second transfer piece having a surface with acavity therein, the second transfer piece being movable relative to thesecond separation piece so that the magnetic particles together with thenucleic acid move out of the first receiving chamber and past thesurface of the second separation piece; and a second receiving chamberwhich receives the magnetic particles and nucleic acid after moving pastthe surface of the second separation piece. The cassette may yet furtherinclude a third separation piece having a surface; a third transferpiece having a surface with a cavity therein, the third transfer piecebeing movable relative to the third separation piece so that themagnetic particles together with the nucleic acid move out of the secondreceiving chamber and past the surface of the third separation piece;and a third receiving chamber which receives the magnetic particles andnucleic acid after moving past the surface of the third separationpiece.

The first receiving chamber may be a washing chamber, and the cassettemay further include a washing solution in the washing chamber. Thesecond receiving chamber may be a washing chamber, and the cassette mayfurther include a washing solution in the washing chamber. The thirdreceiving chamber may be an elution chamber, and the cassette mayfurther include an elution buffer in the elution chamber for separatingthe magnetic particles and the nucleic acid.

A cassette for preparing a sample is disclosed herein. The cassetteincludes at least one mixing chamber for receiving a sample of cells, anenzyme being added in the mixing chamber to break the cells and releasenucleic acid from the cells to create bulk material and nucleic acid inthe bulk material, magnetic particles being added to the mixing chamberto bind with the nucleic acid; a first separation piece having asurface; a first transfer piece having a surface with a cavity therein;a magnet, positionable to attract the magnetic particles together withthe nucleic acid and at least partially separate the nucleic acid fromthe bulk material in the at least one mixing chamber and to transfer themagnetic particles together with the nucleic acid into the cavity in thesurface of the first transfer piece, the first transfer piece beingmovable relative to the first separation piece so that the magneticparticles together with the nucleic acid move out of the mixing chamberand past the surface of the first separation piece; and a firstreceiving chamber which receives the magnetic particles and nucleic acidafter moving past the surface of the first separation piece.

The second piece may be rotatable relative to the first piece. The firstreceiving chamber may be a washing chamber, and the cassette may furtherinclude a washing solution in the washing chamber. The cassette may alsoinclude an elution chamber, and an elution buffer in the elution chamberfor separating the magnetic particles and the nucleic acid. The cassettemay also include a second separation piece having a surface, a secondtransfer piece having a surface with a cavity therein, the secondtransfer piece being movable relative to the second separation piece sothat the magnetic particles together with the nucleic acid move out ofthe first receiving chamber and past the surface of the secondseparation piece, the elution chamber receiving the magnetic particlesand nucleic acid after moving past the surface of the first separationpiece. The second transfer piece may be moveable relative to the secondseparation piece so that the magnetic particles move out of the elutionchamber, leaving the nucleic acid in the elution chamber.

The secondary chamber may be an elution chamber and the cassette mayfurther include an elution buffer in the elution chamber for separatingthe magnetic particles and the nucleic acid. The cassette may alsoinclude a second separation piece having a surface, a second transferpiece having a surface with a cavity therein, the second transfer piecebeing movable relative to the second separation piece so that themagnetic particles together with the nucleic acid move out of the firstreceiving chamber and past the surface of the second separation piece,and a second receiving chamber which receives the magnetic particles andnucleic acid after moving past the surface of the second separationpiece. The cassette may further include a third separation piece havinga surface, a third transfer piece having a surface with a cavitytherein, the third transfer piece being movable relative to the thirdseparation piece so that the magnetic particles together with thenucleic acid move out of the second receiving chamber and past thesurface of the third separation piece, and a third receiving chamberwhich receives the magnetic particles and nucleic acid after moving pastthe surface of the third separation piece. The second receiving chambermay be a washing chamber, and the cassette may further include a washingsolution in the washing chamber. The third receiving chamber may be anelution chamber, and the cassette may further include an elution bufferin the elution chamber for separating the magnetic particles and thenucleic acid.

A cassette for preparing samples is disclosed herein. The cassetteincludes an enclosure, the enclosure comprising a mixing chamber, themixing chamber including an opening for receiving a sample of cellshaving nucleic acid; a plurality of holding chambers having contents,the contents of one of the plurality of holding chambers comprisingmagnetic particles, and the contents of one of the plurality of holdingchambers comprising a proteinase K solution; a plurality of plungers,each of the plurality of plungers corresponding to one of the pluralityof holding chambers, for transferring the contents of the plurality ofholding chambers into the mixing chamber, the proteinase K solutionbreaking up the cells to release the nucleic acid and the nucleic acidbinding to the magnetic particles in the mixing chamber; a first valve,coupled to the mixing chamber, the first valve including a positionablemagnet for attracting the magnetic particles; a washing chamber, coupledto the first valve; a second valve, coupled to the first washingchamber, the second valve including a positionable magnet for attractingthe magnetic particles; and an elution chamber, coupled to the secondvalve, the elution chamber including an opening for removing the nucleicacid from the elution chamber.

One of the plurality of holding chambers may include a binding solutionand one of the plurality of holding chambers may include a lysissolution, and wherein one of the plurality of plungers may transfer thebinding solution into the mixing chamber and wherein one of theplurality of plungers may transfer the lysis solution into the mixingchamber.

A cassette for preparing a sample is disclosed herein. The cassetteincludes a reaction chamber for receiving a sample of cells; a firstholding chamber; an enzyme in the first holding chamber, the enzymebeing transferable into the reaction chamber to break the cells andrelease nucleic acid from the cells to create bulk material and thenucleic acid in the bulk material; a particle chamber; particles to bindwith the nucleic acid in the particle chamber; a second holding chamber;an elution buffer in the second holding chamber to release the nucleicacid from the particles; and an elution chamber for receiving theelution buffer and the released nucleic acid, wherein the elution bufferis transferable from the holding chamber to the reaction chamber,through the particle chamber, and into the elution chamber.

The cassette may include a third holding chamber and a lysis solution inthe third holding chamber, the lysis solution being transferable intothe reaction chamber to solubilize the bulk material.

The cassette may include a fourth holding chamber and a binding solutionin the fourth holding chamber, the binding solution being transferableinto the reaction chamber to bind the nucleic acid to the particles.

The reaction chamber may be aligned with the particle chamber and theelution chamber may be alignable with the particle chamber.

The cassette may include a waste chamber for receiving the enzyme andbulk material.

The cassette may include a plunger to transfer the contents of thereaction chamber through the particle chamber and into the wastechamber.

The cassette may include a plunger to transfer the contents of thereaction chamber through the particle chamber and into the elutionchamber.

The cassette may include a valve in each of the first and second holdingchambers to transfer the contents of each of the first and secondholding chambers into the reaction chamber.

The valve may include a plunger to transfer the contents from the valveinto the reaction chamber.

Another cassette for preparing samples is also disclosed herein. Thecassette includes an enclosure, the enclosure including a reactionchamber, the reaction chamber including an opening for receiving asample of cells having nucleic acid; a plurality of holding chambers; anenzyme in one of the plurality of holding chambers; a lysis buffer inone of the plurality of holding chambers; a binding buffer in one of theplurality of holding chambers; an elution buffer in one of the pluralityof holding chambers; a particle chamber; particles in the particlechamber, the particles to releasably bind with the nucleic acid; and anelution chamber to receive the released nucleic acid, the elutionchamber including an opening for removing the nucleic acid from theenclosure.

The enclosure may include a waste chamber.

Each of the plurality of holding chambers may include a plunger fortransferring contents of the plurality of holding chambers to thereaction chamber.

The cassette may include one or more washing buffers in one or more ofthe plurality of holding chambers.

The reaction chamber may be aligned with the particle chamber andwherein the elution chamber may be alignable with the particle chamber.

A further cassette for preparing a sample is also disclosed herein. Thecassette includes a reaction chamber for receiving a sample of cells; afirst holding chamber; an enzyme in the first holding chamber, theenzyme being transferable into the reaction chamber to break the cellsand release nucleic acid from the cells to create bulk material and thenucleic acid in the bulk material; a particle chamber; particles to bindwith the nucleic acid in the particle chamber; a second holding chamber;an elution buffer in the second holding chamber to release the nucleicacid from the particles; and an elution chamber for receiving theelution buffer and the released nucleic acid, wherein each of theholding chambers comprises an outer housing having a first chambertherein and at least one opening, the first chamber receiving a valve,the valve comprising: an inner housing having a second chamber thereinand at least one opening, the inner housing rotatable relative to theouter housing/the at least one opening of the inner housing alignablewith the at least one opening of the outer housing, the second chamberhaving contents; and a plunger in the second chamber to transfer thecontents of the second chamber through the at least one opening of theinner housing and the at least one opening of the outer housing when theat least one opening of the inner housing and the at least one openingof the outer housing are aligned.

The cassette may include a third holding chamber and a lysis solution inthe third holding chamber, the lysis solution being transferable intothe reaction chamber to solubilize the bulk material.

The cassette may include a fourth holding chamber and a binding solutionin the fourth holding chamber, the binding solution being transferableinto the reaction chamber to bind the nucleic acid to the particles.

The cassette may include a waste chamber for receiving the enzyme andbulk material.

The cassette may include a plunger to transfer the contents of thereaction chamber through the particle chamber and into the wastechamber.

The cassette may include a plunger to transfer the contents of thereaction chamber through the particle chamber and into the elutionchamber.

The reaction chamber may be aligned with the particle chamber andwherein the elution chamber may be alignable with the particle chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described by way of example with reference to theaccompanying drawings, wherein:

FIG. 1 is a cross-sectional side view of a cassette for preparingsamples according to an embodiment of the invention;

FIG. 2 is a perspective view of a cassette for preparing samplesaccording to an embodiment of the invention;

FIG. 3 is a cross-sectional side view showing a sample being placed intothe cassette using a pipette, according to an embodiment of theinvention;

FIG. 4 is a perspective view of a magazine, in which the cassette ofFIG. 1 is used, according to an embodiment of the invention;

FIG. 5 is a perspective view of an instrument, in which the magazine ofFIG. 4 is used, according to an embodiment of the invention;

FIG. 6 is a cross-sectional side view of the cassette of FIG. 1, showingthe transfer of a PK solution into a mixing chamber, according to anembodiment of the invention;

FIG. 7 is a cross-sectional side view of the cassette of FIG. 1, showingthe transfer of a lysis solution into a mixing chamber, according to anembodiment of the invention;

FIG. 8 is a cross-sectional side view of the cassette of FIG. 1, showingthe transfer of a binding solution into a mixing chamber, according toan embodiment of the invention;

FIG. 9 is a cross-sectional side view of the cassette of FIG. 1, showingthe transfer of metallic beads into the mixing chamber, according to anembodiment of the invention;

FIG. 10 is a cross-sectional side view of the cassette of FIG. 1,showing metallic beads bound to a first valve, according to anembodiment of the invention;

FIG. 11 is a cross-sectional side view of the cassette of FIG. 1,showing the transfer of metallic beads from a mixing chamber to awashing chamber, according to an embodiment of the invention;

FIG. 12 is a perspective end view of a valve for use in the cassette ofFIG. 1 according to an embodiment of the invention;

FIG. 13 is a cross-sectional side view of the cassette of FIG. 1,showing metallic beads bound to a second valve, according to anembodiment of the invention;

FIG. 14 is a cross-sectional side view of the cassette of FIG. 1,showing the transfer of metallic beads from a first washing chamber to asecond washing chamber, according to an embodiment of the invention;

FIG. 15 is a cross-sectional side view of the cassette of FIG. 1,showing metallic beads bound to a third valve, according to anembodiment of the invention;

FIG. 16 is a cross-sectional side view of the cassette of FIG. 1,showing the transfer of metallic beads from a second washing chamber toan elution chamber, according to an embodiment of the invention;

FIG. 17 is a cross-sectional side view of the cassette of FIG. 1,showing transfer of metallic beads from an elution chamber to a secondwashing chamber, according to an embodiment of the invention;

FIG. 18 is a cross-sectional side view of the cassette of FIG. 1,showing removal of a prepared sample from an elution chamber, accordingto an embodiment of the invention;

FIG. 19 is a perspective view of a magazine in which a multi-channelpipette is used to access a plurality of samples from a plurality ofcassettes;

FIG. 20 is a cross-sectional perspective view of an alternativeembodiment of the cassette of FIG. 1 according to an embodiment of theinvention;

FIG. 21 is a detailed perspective view of an assembly component of thecassette of FIG. 20 according to an embodiment of the invention;

FIG. 22 is a detailed perspective view of a plunger of the cassette ofFIG. 20 according to an embodiment of the invention;

FIG. 23 is a detailed perspective view of a valve of the cassetteaccording to an embodiment of the invention; and

FIG. 24 is a detailed perspective view of the valve of FIG. 23 accordingto an embodiment of the invention.

FIG. 25 is a perspective view of a cassette for preparing samplesaccording to one embodiment of the invention;

FIG. 26 is a partial cross-sectional view of the cassette of FIG. 25;

FIG. 27 is a partial cross-sectional perspective view of a valve for usein the cassette of FIG. 25;

FIG. 28 is a partial cross-sectional side view of the valve of FIG. 25;

FIG. 29 is a cross-sectional perspective view of the cassette of FIG.25, showing the addition of a sample into a mixing chamber, according toan embodiment of the invention;

FIG. 30 is a cross-sectional perspective view of the cassette of FIG.25, showing the transfer of a PK solution into a mixing chamber,according to an embodiment of the invention;

FIG. 31 is a cross-sectional perspective view of the cassette of FIG.25, showing the transfer of a lysis solution into a mixing chamber,according to an embodiment of the invention;

FIG. 32 is a cross-sectional perspective view of the cassette of FIG.25, showing the transfer of a binding solution into a mixing chamber,according to an embodiment of the invention;

FIG. 33 is a cross-sectional perspective view of the cassette of FIG.25, showing transfer of a sample through particles and into a wastechamber, according to an embodiment of the invention;

FIG. 34 is a cross-sectional perspective view of the cassette of FIG.25, showing the pumping of a wash buffer into the mixing chamber,according to an embodiment of the invention;

FIG. 35 is a cross-sectional perspective view of the cassette of FIG.25, showing the pumping of the wash buffer through the particles,according to an embodiment of the invention;

FIG. 36 is a cross-sectional perspective view of the cassette of FIG.25, showing the pumping of a second wash buffer into the mixing chamber,according to an embodiment of the invention;

FIG. 37 is a cross-sectional perspective view of the cassette of FIG.25, showing the pumping of the second wash buffer through the particles,according to an embodiment of the invention;

FIG. 38 is a cross-sectional perspective view of the cassette of FIG.25, showing the pumping of an elution buffer into the mixing chamber,according to an embodiment of the invention;

FIG. 39 is a cross-sectional perspective view of the cassette of FIG.25, showing the pumping of the elution buffer through the particles andinto the elution chamber, according to an embodiment of the invention;and

FIG. 40 is a cross-sectional perspective view of the cassette of FIG.25, showing the removal of the sample from the elution chamber,according to an embodiment of the invention.

DETAILED DESCRIPTION

FIG. 1 illustrates a cassette 10, which can be used to prepare cellsamples. The cassette 10 includes a housing 12, a mixing chamber 14,first, second third and fourth holding chambers 16, 18,20 and 22, first,second, third and fourth plungers 24,26,28 and 30, first, second andthird valves 32, 34 and 36, first and second washing chambers 38 and 40,an elution chamber 42, first, second, third and fourth pumps 44, 46, 48and 50, first and second lids 52 and 54, first and second heatingelements 56 and 58 and a magnet 60.

Each of the chambers 14, 16, 18, 20, 22, 38, 40 and 42, plungers 24, 26,28 and 30, valves 32, 34 and 36, pumps 44, 46, 48 and 50, and heatingelements 56 and 58 are enclosed within the housing. The lids 52 and 54are movably attached to the housing 12. The magnet 60 is removablypositionable in the first valve 32, second valve 34 and third valve 36.

The mixing chamber 14 has a top surface 62, a bottom surface 64 andopposing side surfaces 66, 68. The top surface 62 of the mixing chamberincludes an opening 70 therein.

The first lid 52 is configured to provide access to the opening 70 inthe top surface 62 of the mixing chamber. The first lid 52 and theopening 70 are coaxial. The first lid 52 is shown being movably attachedto the housing 12, such that when the lid 52 is open or off, the opening70 is accessible and if the lid 52 is closed or on, the opening 70 isnot accessible.

A thin film 74 forms one wall of the mixing chamber 14. The thin film 74is breakable, such that the mixing chamber 14 is accessible when thethin film 74 has been broken or ruptured.

The first holding chamber 16, second holding chamber 18, third holdingchamber 20 and fourth holding chamber 22 are shown located next to themixing chamber 14 and aligned vertically with one another. Each of theholding chambers 16, 18,20, 22 has an opening 76 next to the thin film74 of the mixing chamber 14.

The cassette 10 further includes magnetic iron particles in the form ofmagnetic iron beads in the first holding chamber 16. The cassette 10further includes a binding solution in the second holding chamber 18.The cassette 10 further includes a lysis solution in the third holdingchamber 20. The cassette 10 further includes a proteinase K (PK)solution in the fourth holding chamber 22.

The first, second, third and fourth plungers 24, 26, 28 and 30 arelocated in the first, second, third and fourth holding chambers 16, 18,20 and 22, respectively.

Each of the plungers 16, 18,20,22 includes a base 78, a shaft 80 and apiercing element 82. The shaft 80 extends from the base 78. The piercingelement 82 is at the end of the shaft 80 opposing the base 78 and ispointed. The piercing element 82 is configured to break or rupture thethin film 74 of the mixing chamber 14.

The first pump 44 is a bellows pump having a pumping portion and anozzle portion. The nozzle portion of the first pump 44 is locatedinside the mixing chamber 14. The pumping portion of the first pump 44is located outside the mixing chamber, such that the pumping portion isactuatable.

A heating element 56 is provided at the bottom surface 64 of the mixingchamber 14 for heating the contents of the mixing chamber 14. Theheating element 56 may be a variable heating element.

The opposing side surface 68 of the mixing chamber 14 also includes anopening 84. A first valve 32 is provided between the opening 84 in theside 68 of the mixing chamber 14 and the first washing chamber 38.

The first valve 32 has a first stationary piece 86 and a second moveablepiece 88, the second piece 88 being moveable relative to the first piece86. The first stationary piece 86 includes a first opening 90 and asecond opening 92 and has a surface 94. The second piece 88 has anopening 94 therein for receiving the magnet 60. The second piece 88 hasa surface 96 with a cavity 98 therein. The magnet 60 is shaped tocorrespond to the opening 94 in the second piece 88. The magnet 60 ismoveable in the opening 94 of the second piece 88, and is removable fromthe second piece 88.

The cassette 10 includes a washing solution in the first washing chamber38. The second pump 46 is also a bellows pump, and the nozzle portion ofthe second pump 46 is located in the first washing chamber 38.

The second valve 34 is provided between the first washing chamber 38 andthe second washing chamber 40. The second valve 34 is structurally andfunctionally the same as the first valve 43, and also includes a firststationary piece 86 and a second moveable piece 88. The first stationarypiece 86 includes a first opening 90 and a second opening 92 and has asurface 94. The second moveable piece 88 has a surface 96 with a cavity98 therein.

The cassette 10 includes a washing solution in the second washingchamber 40. The third pump 48 is also a bellows pump, and the nozzleportion of the third pump 48 is located in the second washing chamber40.

The third valve 36 is provided between the second washing chamber 40 andthe elution chamber 42. The third valve 36 is structurally andfunctionally the same as the first valve 32 and the second valve 34, andalso includes a first stationary piece 86 and a second moveable piece88. The first stationary piece 86 includes a first opening 90 and asecond opening 92 and has a surface 94. The second moveable piece 88 hasa surface 96 with a cavity 98 therein.

The cassette 10 includes a washing solution in the elution chamber 42.The fourth pump 50 is also a bellows pump, and the nozzle portion of thefourth pump 50 is located in the elution chamber 42.

A heating element 58 is provided at the bottom surface of the elutionchamber 42 for heating the contents of the elution chamber 42. Theheating element 58 may be a variable heating element.

The elution chamber 42 includes an opening 100 at its top surface foraccessing the contents of the elution chamber 42.

The second lid 54 is configured to provide access to the opening 100 inthe top surface of the elution chamber 42. The second lid 54 is coaxialwith the opening 100. The second lid 54 is shown being movably attachedto the housing 12, such that when the lid 54 is open or off, the opening100 is accessible and if the lid 54 is closed or on, the opening 100 isnot accessible.

With reference to FIG. 2, as described above, the cassette 10 includes ahousing 12. The housing 12 includes a first assembly component 102, asecond assembly component 104 and a third assembly component 106.

The first assembly component 102 includes the mixing chamber 14, thewashing chambers 38 and 40, the elution chamber 42 and the firststationary piece 86 of each of the valves 32, 34 and 36. The firstassembly component 102 also includes attachment parts 108, 110 (seeFIG. 1) at one of its ends and an attachment piece 112 (see FIG. 1).

The second assembly component 104 includes the holding chambers 16, 18,20 and 22 and an opening for receiving the first pump 44. The secondassembly component 104 also includes attachment receiving parts 114, 116(see FIG. 1).

The third assembly component 106 includes openings for receiving thesecond, third and fourth pumps 46, 48 and 50, respectively, and includeslids 52 and 54.

The cassette 10 is assembled by inserting the attachment components 108,110 of the first assembly component 102 into the attachment receivingcomponents 114,116 of the second assembly component 104, respectively.The third assembly component 106 is then secured to the first assemblycomponent using the attachment piece 112, thereby forming the assembledcassette 10, as illustrated in FIG. 2. The plungers 24, 26, 28 and 30,pumps 44, 46, 48 and 50, and the second moveable piece 88 of each of thevalves 32, 34 and 36, are inserted into the cassette 10.

In use, as shown in FIG. 3, the first lid 52 is removed to provideaccess to the opening 70 of the mixing chamber 14. A sample of cells isplaced into an assembled cassette 10 using a pipette 118. The cells inthe sample include nucleic acid. The pipette 118 having the sampletherein is placed in the mixing chamber 14. The sample is released fromthe pipette 118.

As shown in FIG. 4, the cassette 10 is closed by closing the first lid52. The cassette 10 is then placed together with similar cassettes 10into a magazine 120, or rack, for containing a series of cassettes 10.

As shown in FIG. 5, the magazine 120 is placed into an instrument 122. Aprotocol may be selected for preparing the sample in the cassette 10 inthe instrument 122.

As shown in FIG. 6, the PK solution is added to the sample. The PKsolution is added by moving the plunger 30 in the fourth holding chamber22. A force is applied to the base 78 of the plunger 30 to move theplunger 30. As the piercing element 82 of the plunger 30 advances towardthe mixing chamber 14, the piercing element 82 punctures and rupturesthe thin film 74. The break in the thin film 74 provides access to themixing chamber 14. Continued motion of the plunger 30 transfers thecontents (e.g., PK solution) of the first holding chamber 22 into themixing chamber 14.

The PK solution is mixed with the sample by pumping the mixture with thefirst pump 44. The PK solution destroys the walls of the cells of thesample, creating bulk material and nucleic acid in the bulk material.

As shown in FIG. 7, the lysis solution is added to the sample. Plunger28 operates in the same manner as plunger 30 to transfer the lysissolution in the third holding chamber 20 into the mixing chamber 14. Thesample is pumped to mix the lysis buffer with the PK solution and sampleof cells. The lysis solution is typically a salt or detergent. The lysissolution is used to solulibize the bulk material. The lysis solutiontypically does not solulibize proteins.

The heating element 56 may be used to heat the lysis solution andsample. The heating element 56 may be controlled by the instrument 122.As described hereinabove, the temperature of the heating element 56 maybe variable, and is selected to optimize the effectiveness of the lysissolution.

As shown in FIG. 8, the binding solution is added to the sample, PKsolution and lysis buffer solution. Plunger 26 operates in the samemanner as plunger 30 to transfer the binding solution in the secondholding chamber 18 into the mixing chamber 14. The solution is pumped tomix the binding solution with the PK solution, lysis solution andsample. The binding solution is typically hydrophobic and increases saltin the solution. The binding solution causes the nucleic acid to bemagnetically charged.

As shown in FIG. 9, the magnetic beads are added to the solution andpumped for about two minutes. Plunger 24 operates in the same manner asplunger 30 to transfer the lysis solution in the first holding chamber18 into the mixing chamber 14. The magnetic beads bind to themagnetically charged nucleic acid.

As shown in FIG. 10, the magnetic beads, together with the nucleic acid,are bound to the first valve 32. The removable positionable magnet 60 isplaced in the first valve 32 and slid to a position in the first valve32 to attract the magnetic beads, which are bound to the nucleic acid,from the mixing chamber 14 to the first valve 32.

As shown in FIG. 11, the magnetic beads, together with the nucleic acid,are then moved from the mixing chamber 14 and received in the firstwashing chamber 38.

FIG. 12 is a detailed view of the valves 32, 34, 36 illustrating themovement of the magnetic beads from the mixing chamber 14 to the firstwashing chamber 38. As discussed above each of the valves 32, 34 and 36include a first stationary piece 86 and a second moveable piece 88, thesecond piece 88 being moveable relative to the first piece 86.

The magnet 60 is inserted into the opening 94 of the second piece 88.The magnet 60 is inserted to a position corresponding to the openings 90and 92 of the first piece 86. The magnet 60 attracts the magnetic beadsfrom the mixing chamber 14 through the opening 90 in the first piece 86and into the cavity 98 in the second piece 88. The second piece 88 isrotated such that the magnetic beads are sealed in the cavity 98 of thesecond piece 88, between surfaces of the second piece 88 and the firstpiece 86. The second piece 88 is rotated past the surface 94 of thefirst piece 86, such that the cavity 98 is accessible in the opening 92of the first piece 86. The magnet 60 is then removed from the opening 94in the second piece 88 to release the magnetic beads from the cavity 98in the second piece 88.

As shown in FIG. 13, the magnetic beads and nucleic acid are then washedwith the washing solution by pumping the solution with the second pump46. The magnetic beads, together with the nucleic acid, are then boundto the second valve 34 by inserting the magnet 60 into the second valve34, as described above with reference to FIG. 12.

As shown in FIG. 14, the magnetic beads, together with the nucleic acid,are then moved from the first washing chamber 38 to the second washingchamber 40 using the second valve 34. The second valve 34 transfers themagnetic beads and nucleic acid from the first washing chamber 38 to thesecond washing chamber 40, as described above with reference to FIG. 12.

As shown in FIG. 15, the magnetic beads and nucleic acid are then washedwith the washing solution a second time by pumping the solution with thethird pump 48. The magnetic beads, together with the nucleic acid, arethen bound to the third valve 36 by positioning the magnet 60 in thethird valve 36, as described above with reference to FIG. 12.

As shown in FIG. 16, the magnetic beads and nucleic acid are then movedfrom the second washing chamber 40 to the elution chamber 42. Themagnetic beads and nucleic acid are transferred from the second washingchamber 40 to the elution chamber 42 using the procedure described abovewith reference to FIG. 12.

An elution buffer solution is then mixed with the magnetic beads andnucleic acid by pumping the solution with the fourth pump 50. Theheating element 58 may be used to heat the elution buffer, magneticbeads and nucleic acid. The heating element 58 may be controlled by theinstrument 122. The temperature may be variable and may be selected tooptimize release of the nucleic acid from the magnetic beads.

The magnetic beads alone are then bound again to the third valve 36 bypositioning the magnet 60 in the third valve 36 as described above withreference to FIG. 12.

As shown in FIG. 17, the magnetic beads alone are then moved from theelution chamber 42 back into the second washing chamber 40, leaving thenucleic acid in the elution chamber 42. The magnetic beads aretransferred from the elution chamber 42 to the second washing chamber 40using the procedure described above with reference to FIG. 12.

As shown in FIG. 18, the prepared sample of nucleic acid may be accessedusing a second pipette 124. The second lid 54 is removed to provideaccess to the opening 100 in the elution chamber 42. The pipette 124 isinserted into the opening 100 and the prepared sample of nucleic acid iswithdrawn.

As shown in FIG. 19, a multi-channel pipette 126 may be used to access aplurality of samples from a plurality of cassettes 10.

FIG. 20 illustrates an alternative embodiment of the cassette 10. Thecassette 10 a illustrated in FIG. 20 differs from the cassette 10illustrated in FIG. 1 in that the assembly component 104 a includes aseal 130, the plungers 24 a, 26 a, 28 a and 30 a each include seals 132,134, 136 and 138, respectively, and the valves 32 a, 34 a and 36 a havea different arrangement, as discussed hereinafter.

FIG. 21 illustrates the assembly component 104 a in more detail. Theassembly component 104 a includes a seal 130. The illustrated seal 130is a double elastomer, which extends along the circumference of theassembly component 104 a.

FIG. 22 illustrates the plunger 24 a in more detail. The plunger 24 aincludes a seal 132. The illustrated seal 132 is also a doubleelastomer, which extends along the circumference of the plunger 24 a. Itwill be appreciated that each of plungers 26 a, 28 a and 30 a may alsohave a similar arrangement.

FIGS. 23 and 24 illustrate the valve 32 a in more detail. It will beappreciated that valves 34 a and 36 a also have a similar arrangement.The valve 32 a includes a magnet 60 a, a housing 142, and a shaft 144.The housing 142 includes a first opening (not shown) to receive themagnet 60 a and a second opening 148 to expose the magnet 60 a andreceive the particles 146. The magnet 60 a is shaped to correspond tothe opening 148 and is selected to attract the particles 146. Thehousing 142 also includes a third opening (not shown) for receiving theshaft 144. As shown in FIG. 24, the shaft 144 may include a keyedelement 150. The keyed element 150 is shaped to engage the cassette 10a. It will be appreciated that the shaft may be removable or anintegrated element of the valve 32 a. It will also be appreciated thatthe housing 142 may, alternatively, include the keyed element.

The shaft 144 is engageable with the housing 142 and magnet 60 a torotate the housing 142 and magnet 60 a relative to the cassette 10 a tomove the particles 146 from the mixing chamber 14 a to the washingchamber 38 a. It will be appreciated that valves 34 a and 36 a operatein a similar manner to transfer the particles 146 from the washingchamber 38 a to the washing chamber 40 a and from the washing chamber 40a to the elution chamber 42 a, respectively.

In one embodiment, a total of about 200 μL sample is placed into thecassette. The sample is mixed with a total of about 50 μL of the PKsolution by pumping the mixture of the sample and PK solution for aboutone minute. A total of about 200 μL of the lysis solution is added tothe sample and PK solution, and the solutions are pumped for about oneminute to mix the solutions. The mixture is then heated at about 60° C.for about ten minutes, and the mixture is allowed to cool for about 5minutes. The mixture is further pumped while it cools. A total of about500 μL of binding solution is added to the mixture. The solutions arepumped for about one minute. The magnetic beads are added to thesolution and pumped for about two minutes. The magnetic beads aretransferred and washed as described above. A total of about 700 μL ofwashing solution is provided in each of the washing chambers. A total ofabout 200 μL of elution solution is provided in the elution chamber. Themagnetic beads are mixed with the elution solution by pumping themixture for about one minute. The mixture is then heated at about 90° C.for about two minutes. The process continues as previously described.

Although the cassette 10 has been described as having a mixing chamber14, two washing chambers 38 and 40 and an elution chamber 42, it isenvisioned that only one washing chamber or no washing chamber mayalternatively be provided.

Although the cassette has been described as using a single removablemagnet 60, it is envisioned that each valve may include a positionablemagnet, such that the magnet does not need to be removed. The magnet 60may be rotatable, and used to rotate the second piece of the valves.Alternatively, the magnet may only slide inside of each of the valves,and the second piece is rotated independent of the magnet.

It is envisioned that a cassette 10 that does not use valves asdescribed herein may be used to transfer the magnetic particles from themixing chamber to the elution chamber. In such an embodiment, aslideable magnet may be provided to transfer the magnetic particles fromone chamber to the next.

Although the cassette 10 has been described as using a PK solution,lysis solution, binding solution and magnetic beads to release thenucleic acid and magnetic beads, it is envisioned that it may bepossible to practice the invention without using each of the abovesolutions. In addition, although the solution was described as using aPK solution to break up the cells, it is envisioned that any enzymewhich causes cells to break up to release nucleic acid may be used withthe invention.

It is envisioned that the housing 12 may be transparent, such that theprocedure can be viewed.

In one embodiment the thin film 74 is a lamination.

In one embodiment, the lids 52 and 54 may be screw-top lids. In oneembodiment, the lids 52, 54 include a hydrophobic membrane, which allowsgasses to vent through the lid, but does not allow the liquids to escapethe cassette 100.

In one embodiment, pump 50 is insertable into opening 100. In oneembodiment, pump 50 can also be used as a pipette to remove the samplefrom the cassette 10.

It is also envisioned that the mixing chamber 14 may be provided withouta puncturable thin film 74. In such an embodiment, the plungers 24, 26,28 and 30 would not need a piercing element 82. Instead, the plungers24, 26, 28 and 30 would have a sealing element to prevent leakage of thecontents of the holding chamber 16, 18, 20 and 22, associated with eachplunger 24, 26, 28 and 30, respectively, until the plunger was moved.

FIG. 25 illustrates a cassette 200, which can be used to prepare cellsamples. The cassette 200 includes a housing 202, first, second, third,fourth, fifth, sixth, seventh and eighth holding chambers 204 a-h,respectively. Each of the holding chambers 204 a-h includes a valveassembly 206 a-h therein. A locking element 207 may also be provided.

FIG. 26 illustrates the cassette 200 in more detail. The cassette 200further includes a reaction chamber 208, a particle chamber 210, a wastechamber 212, a waste overflow chamber 214, an elution chamber 216, aplunger 218, and first and second lids 220 and 222, respectively. Thecassette 200 may also include one or more heating elements (not shown).

Each of the holding chambers 204 a-h, valve assemblies 206 a-h, reactionchamber 208, particle chamber 210, waster chamber 212, waster overflowchamber 214, and plunger 218 are enclosed within the housing 202. Thelids 222, 224 are movably or removably attached to the housing 202.

The reaction chamber 208 has a top surface 226, a bottom surface 228 andopposing side surfaces 230, 232.

The top surface 226 of the reaction chamber 208 includes an opening 234therein. The first lid 222 is configured to provide access to theopening 234 in the top surface 226 of the reaction chamber 208. Theillustrated lid 222 is a screw-top lid; however, any other lid which(removably) provides access to the opening 234.

The bottom surface 228 of the reaction chamber 208 includes an opening236 therein. The opening 236 allows the reaction chamber 208 to be influid communication with the particle chamber 210.

The side surface 232 includes openings 238 a-h therein. The openings 238ah allow the reaction chamber 208 to be in fluid communication with theholding chambers 204 a-h, respectively.

The cassette 200 includes a binding solution in a holding chamber 204 a.The cassette 200 further includes a lysis solution in a holding chamber204 b. The cassette 200 further includes a proteinase K (PK) solution ina holding chamber 204 c. The cassette 200 further includes a washingsolution in one or more of the holding chambers 204 d-e. The cassette200 further includes an elution solution in a holding chamber 204 f.

The plunger 218 and the first lid 222 are shown attached to one anotherto form an integral plunging system. The plunger 218 is compressible topump the contents of the reaction chamber 208. Alternatively, a separatepump may also be provided to pump the contents of the reaction chamber208. The plunger 218 is also moveable within the reaction chamber 208 topush the contents of the reaction chamber 208 through the particlechamber 210.

The holding chambers 204 a-h are formed in the housing 202 of thecassette 200. Each of the holding chambers 204 a-h include a guide 240a-h engageable with a corresponding slot in the valve assembly 206. Theholding chambers 204 a-h also include at least one opening 242 a-h,engageable with corresponding openings in the valve assembly 206 a-h.The housing 202 also includes slots 244 a-h, engageable withcorresponding guides in the valve assembly 206 a-h.

The particle chamber 210 includes a body 250, having a first opening252, a second opening 254, and a plurality of particles 256 thereon. Theparticles may be magnetic or nonmagnetic, depending on the applicationof the cassette 200. The particles may be, for example, cellulose,plastic or iron. The particle chamber 210 is shown aligned with thereaction chamber 208.

The waste chamber 212 and the elution chamber 216 are integrated withone another and are rotatable relative to the housing 202. The wasteoverflow chamber 214 is positioned near the waste chamber 212 and iscapable of being in fluid communication with the waste chamber 212. Thewaste chamber 212 and elution chamber 216 are alignable with theparticle chamber and are capable of being in fluid communication withthe particle chamber 210.

The waste chamber 212 has a top surface 270, a bottom surface 272, aninner surface 274 and an outer surface 276. The overflow waste chamber214 has a top surface 278, a bottom surface 280 and opposing sidesurfaces 282, 284. The elution chamber 216 also has a top surface 286, abottom surface 288, an inner surface 290 and an outer surface 292.

It will be appreciated that the outer surface 276 of the waste chamber212 and the outer surface 292 of the elution chamber 216 are integratedwith one another. It will also be appreciated that the inner surface 274of the waste chamber 212 is the same as the inner surface 290 of theelution chamber 216.

The top surface 270 of the waste chamber 212 and the top surface 286 ofthe elution chamber 216 each have an opening 294, 296, respectively.These openings 294,296 are alignable with the opening 252 in theparticle chamber 210 to provide a fluid communication route between theparticle chamber 210 and the waste chamber 212 and the elution chamber216.

The outer surface 276 of the waste chamber 212 includes an opening 298therein. One of the side surfaces 282,284 of the overflow waste chamber214 includes an opening 300 therein. The opening 298 and opening 300 arealignable, such that fluid flowing into the waste chamber 212 can flowfrom the waste chamber 212 and into the overflow waste chamber 214.

The bottom surface 288 of the elution chamber 216 includes an opening302 therein. The second lid 224 is configured to provide access to theopening 302 in the bottom surface 288 of the elution chamber 216. Theillustrated lid 224 is a screw-top lid; however, any other lid which(removably) provides access to the opening 302.

FIGS. 27 and 28 illustrate the valve assembly 206 in more detail. Valveassembly 206 includes a housing 310. The housing 310 includes a chamber312 therein, a slot 314, and projections 316 extending therefrom. Thechamber 312 includes a pump 318 therein. A lid 320 is provided at an endof the housing to seal the chamber 310. The lid includes first andsecond openings 322, 324, extending therethrough and providing fluidcommunication with the chamber 312.

When the openings 322, 324 are not aligned with openings 238 a-h, thecontents of the chamber 312 are sealed within the valve assembly;however, when the openings 322, 324 are aligned with openings 238 a-h,the contents of the chamber 312 are releasable from the chamber 312. Theslot 314 is used to guide the openings 322,324 to the location where theopenings 322, 324 are aligned with the openings 238 a-h. The pump 318 isused to transfer the contents from the chamber 312 and into the reactionchamber 214 through the openings 322, 324.

The cassette 200 is assembled by inserting the valve assemblies 206 a-hinto the holding chambers 204 a-h of the housing. The locking element207 may be connected to the housing to secure valve assemblies 206 a-hin the holding chambers 204 a-h. The waste chamber and elution chamber216 assembly is inserted into the housing 202 and the lid 224 is securedto the housing 202. The plunger 218 is also inserted into the reactionchamber 208 and the lid 222 is secured to the housing.

In use, as shown in FIG. 29, the first lid 222 is removed to provideaccess to the opening 234 of the reaction chamber 208. A sample of cells350 is placed into the cassette 200 using a pipette 352. The cells inthe sample include nucleic acid. The pipette 352 having the sampletherein is placed in the reaction chamber 208. The sample is releasedfrom the pipette 352.

As shown in FIG. 30, the PK solution is added to the sample. The PKsolution is added by rotating the valve assembly 206 c relative to thehousing 202. At least one of the openings 322, 324 of the valve assembly206 c is aligned with the opening 242 c in the holding chamber 204 c torelease the PK solution from the holding chamber 204 c and into thereaction chamber 208 through the opening 238 c in the housing 202.

The PK Solution is mixed with the sample by pumping the mixture with theplunger 218. As described hereinabove, the PK solution destroys the wallso the cells of the sample, creating bulk material and nucleic acid inthe bulk material.

As shown in FIG. 31, the lysis solution is added to the sample. Thevalve assembly 206 b operates in the same manner as valve assembly 206 cto transfer the lysis solution in the holding chamber 204 b into thereaction chamber 208. The sample is typically pumped to mix the lysissolution with the mixed PK solution and sample. The lysis solution istypically a salt or detergent, and is used to solulibize the bulkmaterial, as discussed hereinabove.

As shown in FIG. 32, a binding solution is added to the sample, PKsolution and lysis solution. Valve assembly 206 a operates in the samemanner as valve assembly 206 c to transfer the binding solution in theholding chamber 204 a into the reaction chamber 208. The solution ispumped to mix the binding solution with the PK solution, lysis solutionand sample. The binding solution is typically hydrophobic and increasessale in the solution.

As shown in FIG. 33, the solution is pumped through the particles in theparticle chamber 210. The nucleic acid binds to the particles in theparticle chamber, while the remaining solution flows into the wastechamber 212, and, if needed, the waste overflow chamber 214. Asdescribed above, when the opening 298 in the waste chamber 212 isaligned with the opening 300 in the waste overflow chamber 214, thesolution can flow into the waste overflow chamber 214 from the wastechamber 212.

As shown in FIG. 34, the washing solution is added to the reactionchamber 208 by operating the valve assembly 206 d in the same manner asvalve assembly 206 c to transfer the washing solution from the holdingchamber 204 d into the reaction chamber 208.

As shown in FIG. 35, the first washing solution is pumped through theparticle chamber 210 and into the waste chamber 212, and, if needed, thewaste overflow chamber 214.

As shown in FIG. 36, a second washing solution is added to the reactionchamber 208 by operating the valve assembly 206 e in the same manner asvalve assembly 206 c to transfer the washing solution from the holdingchamber 204 e into the reaction chamber 208.

As shown in FIG. 37, the second washing solution is pumped through theparticle chamber 210 and into the waste chamber 212, and, if needed, thewaste overflow chamber 214.

As shown in FIG. 38, an elution solution is added to the reactionchamber 208 by operating the valve assembly 206 f in the same manner asvalve assembly 206 c to transfer the elution solution from the holdingchamber 204 f into the reaction chamber 208.

The waste chamber and elution chamber assembly is rotated to align theopening 296 of the elution chamber 216 with the opening 252 of theparticle chamber 210 such that the elution solution is transferable intothe elution chamber 216. It will be appreciated that the opening 296 canbe aligned with the opening 252 before or after the elution solution isadded to the reaction chamber 208.

As shown in FIG. 39, the elution solution is pumped through the particlechamber 210 to elute the bound nucleic acid. The nucleic acid andelution solution flow into the elution chamber 216.

As shown in FIG. 40, the prepared sample of nucleic acid may be accessedusing a pipette 354. The second lid 224 is removed to provide access tothe opening 302 in the elution chamber 216. The pipette 354 is insertedinto the opening 302 and the prepared sample of nucleic acid iswithdrawn.

It will be appreciated that the cassette 200 can be placed with similarcassettes into a magazine or rack for containing a series of cassettes.The magazine or rack can be placed into an instrument, and a protocolmay be selected for preparing the sample in the cassette 200 in theinstrument.

Cassette 200 may include one or more heating elements as describedhereinabove with respect to cassette 100.

The cassettes 100, 200 may be disposable.

It will be appreciated that although the cassettes 100, 200 have beendescribed with respect to breaking cells to extract nucleic acid, thecassettes 100, 200 can be used to break cells to extract other cellcomponents, such as, for example, protein. Also, although a lysissolution has been described as being used to break up cells, it will beappreciated that any substance that can break up cells, such as, forexample, reagents, enzymes, catatropic salts, other lysis solutions andthe like.

The cassettes described herein are advantageous because it is closed.There is no contamination of the sample during the process. In addition,a fewer number of samples, including as few as one sample, may beprepared.

The foregoing description with attached drawings is only illustrative ofpossible embodiments of the described method and should only beconstrued as such. Other persons of ordinary skill in the art willrealize that many other specific embodiments are possible that fallwithin the scope and spirit of the present idea. The scope of theinvention is indicated by the following claims rather than by theforegoing description. Any and all modifications which come within themeaning and range of equivalency of the following claims are to beconsidered within their scope.

What is claimed is:
 1. An apparatus comprising: a first chamber; a firstvalve at least partially disposed in the first chamber, the first valvecomprising a valve housing defining a first valve opening in fluidcommunication with the first chamber and a second valve opening in fluidcommunication with a second chamber, a second valve at least partiallydisposed in the first chamber, the second valve comprising a valvehousing defining a first valve opening in fluid communication with thefirst chamber and a second valve opening in fluid communication with athird chamber, and a pump comprising an actuator and a nozzle, whereinthe actuator is disposed outside of the first chamber and the nozzle isdisposed at least partially inside the first chamber, wherein the pumpis a first pump, and the second chamber comprises a second pumpcomprising an actuator and nozzle, and wherein the actuator is disposedoutside of the second chamber and the nozzle is disposed at leastpartially inside the second chamber.
 2. The apparatus of claim 1,wherein the first chamber is a washing chamber, and the washing chambercontains a washing solution.
 3. The apparatus of claim 1, wherein thesecond chamber is a mixing chamber, and the mixing chamber comprises anopening configured for receiving a sample into the mixing chamber, and aremovable lid configured to provide access to the opening.
 4. Theapparatus of claim 1, wherein the third chamber comprises third pumpcomprising an actuator and a nozzle, wherein the actuator is disposedoutside of the third chamber and the nozzle is disposed at leastpartially inside the third chamber.
 5. The apparatus of claim 1, whereinthe pump is a first pump, and the third chamber comprises a second pumpcomprising an actuator and a nozzle, wherein the actuator is disposedoutside of the third chamber and the nozzle is disposed at leastpartially inside the third chamber.
 6. The apparatus of claim 1, furthercomprising a plurality of reagent chambers, wherein each of theplurality of reagent chambers comprises a puncturable or sealable memberdefining at least a portion of a boundary between the plurality ofreagent chambers and the second chamber.
 7. The apparatus of claim 6,wherein each of the plurality of reagent chambers comprises apuncturable member, and the apparatus further comprises a plunger atleast partially disposed in each of the plurality of reagent chambersand configured to puncture the puncturable member when the plunger ismoved.
 8. The apparatus of claim 6, further comprising a plunger atleast partially disposed in each of the plurality of reagent chambers,the plungers comprising a sealable member configured to prevent leakagebetween the reagent chambers and the second chamber until the plungersare moved.
 9. The apparatus of claim 6, wherein the plurality of reagentchambers contain one or more of a lysis buffer, enzyme, magneticpanicles, or chaotropic salt.
 10. The apparatus of claim 1, wherein thepump is a bellows pump.
 11. The apparatus of claim 3, wherein the mixingchamber contains a sample of cells.
 12. An apparatus for preparing asample of cells, the apparatus comprising: a mixing chamber comprisingan opening configured for receiving a sample of cells into the mixingchamber, and a removable lid configured to provide access to theopening; a first washing chamber; a first valve comprising a valvehousing defining a first valve opening in fluid communication with themixing chamber and a second valve opening in fluid communication withthe first washing chamber; a second valve comprising a valve housingdefining a first valve opening in fluid communication with the firstwashing chamber and a second valve opening in fluid communication with asecond washing chamber or an elution chamber; and a plurality of reagentchambers, where in each of the plurality of reagent chambers comprises apuncturable or sealable member defining at least a portion of a boundarybetween the plurality of reagent chambers and the mixing chamber,wherein the plurality of reagent chambers contain one or more of a lysisbuffer, enzyme, magnetic particles, or chaotropic salt; and a pumpcomprising an actuator and a nozzle, wherein the actuator is disposedoutside of the first chamber and the nozzle is disposed at leastpartially inside the first chamber inside the first chamber, wherein thepump is a first pump, and the second chamber comprises a second pumpcomprising an actuator and nozzle, and wherein the actuator is disposedoutside of the first washing chamber and the nozzle is disposed at leastpartially inside the first washing chamber.
 13. The apparatus of claim12, wherein each of the plurality of reagent chambers comprises apuncturable member, and the apparatus further comprises a plunger atleast partially disposed in each of the plurality of reagent chambersand configured to puncture the puncturable member when the plunger ismoved.
 14. The apparatus of claim 12, further comprising a plunger atleast partially disposed in each of the plurality of reagent chambers,the plungers comprising a sealable member configured to prevent leakagebetween the reagent chambers and the second chamber until the plungersare moved.
 15. The apparatus of claim 12, wherein the pump is a firstpump, the apparatus further comprising a second pump comprising anactuator and a nozzle, wherein the actuator is disposed outside of thefirst second washing chamber or the elution chamber and the nozzle isdisposed at least partially inside the first second washing chamber orthe elution chamber.
 16. The apparatus of claim 12 further comprising asecond washing chamber and an elution chamber.
 17. The apparatus ofclaim 16, further comprising a first pump, a second pump, and a thirdpump, wherein each of the pumps comprises an actuator and a nozzle, andfurther wherein the actuator of the first pump is disposed outside ofthe first washing chamber and the nozzle is disposed at least partiallyinside the first washing chamber, the actuator of the second pump isdisposed outside of the second washing chamber and the nozzle isdisposed at least partially inside the second washing chamber, and theactuator of the third pump is disposed outside of the elution chamberand the nozzle is disposed at least partially inside the elutionchamber.
 18. The apparatus of claim 12, further comprising a pumpcoupled to the mixing chamber, the pump configured to mix a sample ofcells and one or more reagents in the mixing chamber.